1. Field of the Invention
The present invention relates to a method for the amplification of mRNA of a sample, comprising the steps of i.) generating cDNA from polyadenylated RNA employing at least one primer hybridizing to said polyadenylated RNA and comprising a 5′ poly(C) or a 5′ poly(G) flank; ii.)(aa) if present, removing non-hybridized, surplus primer(s) and/or surplus dNTPs; ii.)(ab) 3′ tailing of said generated cDNA with a poly(G) tail when in step i. (a) primer(s) comprising a 5′ poly(C) flank was employed or a poly(C) tail when in step i. (a) primer(s) comprising a 5′ poly(G) flank was employed; or ii.)(b) 3′ tailing of said generated cDNA with a poly(G) tail when in step i. (a) primer(s) comprising a 5′ poly(C) flank was employed or a poly(C) tail when in step i. (a) primer(s) comprising a 5′ poly(G) flank was employed using an RNA-ligase, irrespective of the presence or absence of surplus primer(s) and/or surplus dNTPs; and iii.) amplifying the tailed cDNA with a primer hybridizing to the tail(s) generated in step ii(ab) or ii(b). Furthermore, the present invention relates to methods for the preparation of in vitro surrogate(s), for identifying expressed genes in a test sample, for identifying a drug candidate for therapy of a pathological condition and for in vitro detection of a pathological condition employing said method for amplification of mRNA. In addition, the present invention relates to the use of amplified cDNA(s) as obtained by the method of the invention in hybridization, interaction and/or enzymatic arrays.
2. Description of the Related Art
Several documents are cited throughout the text of this specification. The disclosure content of each of the documents (including any manufacturer's specifications, instructions, etc.) is herewith incorporated by reference.
The study of gene expression and gene expression patterns have lately been revolutionized by global analysis of mRNA expression on cDNA filter assays or cDNA micro arrays (see, inter alia, Southern, Trends Genet. 12 (1996), 110–115; Debouck, Nat. Genet. 21:48–50 (1999); Hacia, Nat. Genet., 21, 42–7 (1999); Cole, Nat. Genet. 21, 38–41 (1999); Bowtell D D., Nat. Genet., 21, 25–32 (1999); Cheung, Nat. Genet., 21, 15–19 (1999); Duggan, Nat. Genet., 21, 10–14 (1999); Southern, Nat. Genet., 21, 5–9 (1999)). For example, Lockhart (Nature Biotechnology 14 (1996), 1675–1680) describes an approach that is based on hybridization of a large number of mRNAs to small, high-density arrays containing tens of thousands of synthetic oligonucleotides, allowing for the simultaneous monitoring of tens of thousands of (expressed) genes. Further micro arrays for gene expression have been described in Shalon (Pathol. Biol. 46 (1998), 107–109), Lockhardt (Nuc. Acids Symp. Ser. 38 (1998), 11–12) or in Schena (Trends Biotech. 16 (1998), 301–306). However, one of the major draw-backs of the above described cDNA-array technology is the fact that these technologies require an amount of 2.5 to 10 μg of nucleic acid probes to be tested either in the form of mRNA, reverse transcribed RNA or amplified cDNA (see, inter alia, Schena (Science 270 (1995), 467–470 and PNAS U.S.A. 93 (1996), 10614–10619) or Lockhardt (1996) loc. cit.). This amount of material is normally only derived from a large of number of cells such as about 109. Bryant, PNAS U.S.A. 96 (1999), 5559–5564 or Mahadevappa, Nat. Biotech. 17 (1999), 1134–1136 reported such an approach using at least from 50000 cells. The smallest number of cells yet used for ex-vivo tissue analysis and corresponding gene expression has been 1,000 cells (Luo, Nat. Medicine 5 (1999), 117–122). However, a plethora of physiological and/or pathological conditions would require to study the gene expression pattern or “transcriptome”, defined as the entirety of mRNA molecules in a given biological sample (Velculescu, Cell, 88, 243–251 (1997) of a lower number of cells or even a single cell. For instance, the investigation of spatially and temporally regulated gene expression in embryogenesis would clearly profit from a method were a low number of cells, in particular a single cell, can be deduced. Similarly, it would be of high interest to investigate the gene expression pattern/transcriptome of individual cells or a low number of cells derived from adult tissue, like, inter alia, blood or neuronal (stem) cells. Furthermore, multiple pathological conditions could be clarified, e.g., the delineation of deregulated gene expression in a typical proliferation, mutaplasia, preneoplastic lesians and/or carcinomata in situ. Other examples of locally restricted pathological processes which could be investigated comprise, but are not limited to, restenosis, Alzheimer's disease, Parkinson's disease, graft-versus-host disease or inflammations in autoimmunity. Furthermore, occult micrometastasis derived from a small cancer has dire consequences if the disseminated tumor cells survive in distant organs and grow into manifest metastases. Tumor cells left after resection of primary tumors are currently detected in bone marrow aspirates by immunocytochemical staining with antibodies directed against cytokeratins (reviewed in Pantel, J. Natl. Canc. Inst. 91, 1113–1124 (1999)). While several studies have established the prognostic significance of cytokeratin-positive micrometastatic cells in bone marrow (Braun, N. Engl. J. Med. 342, 525–533 (2000); Pantel, J. Natl. Canc. Inst. 91, 1113–1124 (1999)), the biology of these cells has largely remained enigmatic because of their extremely low frequency in the range of 10−5–10−6.
The systemic spread of cancer cells requires that cells evade from the solid tumor, distribute via blood or lymphatic vessels, cross endothelial and tissue barriers and survive ectopically as single cells. The phenotypic changes accompanying these steps are considered a developmental process, the so-called epithelial-mesenchymal transformation (EMT) (Hay, Acta Anatomica, 154, 8–20, (1995); Birchmeier, Acta Anatomica, 156, 217–226 (1996)). Only a small fraction of cells disseminated from a tumor may acquire EMT-associated features (Boyer, Acta Anatomica, 156, 227–239 (1996)). The epigenetic changes leading to EMT are not known so far but may have important implications for the development of future therapies.
Major technical hurdles in studying epigenetic changes of, e.g., disseminated tumor cells or pathological modified tissue are limited accessibility, low frequency, unambiguous identification, and subsequent transcriptome analysis at a single cell level or of a low number of cells. A variety of protocols has been developed for the generation of “single cell cDNA libraries” and the global amplification of mRNA from individual cells (see Belyavsky, Nucl. Acid. Res., 17, 2919–2932 (1989); Brady, Methods in Enzymology, 225, 611–623 (1993); and Karrer, Proc. Natl. Acad. Sci. USA, 92, 3814–3818 (1995)). However, these procedures have obvious drawbacks, such as the restriction to 3′-ends and an insufficient sensitivity when PCR amplificates are hybridized to cDNA arrays.
In these procedures, variation introduced during amplification of cDNA fragments was reduced by limiting the length of the cDNAs during reverse-transcription. This was accomplished through low substrate conditions for the reverse-transcriptase; i.e. the use of low concentrations of an oligo d(T) primer and low dNTP concentrations. However, there is a risk of compromising reverse-transcription and subsequent PCR-efficiency which may lead to arbitrary results when transcriptome/gene expression patterns of cells/single cells are to be investigated. Furthermore, the use of an oligo(dT) primer for PCR amplification limits the use of high annealing temperatures and thus stringent annealing conditions. Typically, annealing is performed at 42° C. (Brail, Mut. Res. Genomics 406 (1999), 45–54). As pointed out hereinabove, such an approach may be suitable for a 3′ restricted cDNA synthesis. However, higher annealing temperatures reduce the presence of secondary structures in the cDNA and the likelihood of unspecific annealing to internal sequences of the cDNA, which would result in shortening of the amplificates compared to the cDNA molecules. Annealing temperatures of the method of the invention are preferably above 45° C., more preferably above 55° C., even more preferably above 65° C.
As mentioned hereinabove, the amount of mRNA in a low number of cells or even a single cell is insufficient for use in direct global analysis. Therefore, global analysis of expressed mRNA (of a “transcriptome”) from a low number of cells or even an individual, single cell requires amplification of extracted and/or reverse transcribed polyadenylated mRNA. To date, PCR amplification of small amounts of mRNA has not resulted in reliable representation of the relative expression of mRNA present in a certain cell/low number of cells at a specific timepoint, a specific developmental state and/or a specific physiological state (Brail, Mut. Res. Genomics 406 (1999), 45–54), Brail (1999), (loc. cit.) conclude that the method as described by Brady (Brady (1993) (loc. cit.) is likely to introduce variation(s) in the tailing reaction or the PCR amplification steps. In particular, Brail's analysis (Brail (1999), loc. cit) showed a five-fold variation even for highly-abundant house-keeping genes (direct comparison of GAPDH and ribosomal gene L32).
Thus, the technical problem of the present invention consists in providing means and methods which comply with the need of a global and uniform amplification of mRNA, in particular of the transcriptome of a low number of cells or a single cell. The solution to this technical problem is achieved by providing the embodiments characterized in the claims.